2/12-2/18: Week 6 Tetracycline vs Caeni efflux pump experiment

 Introduction

In lab this week, my group and I did one more testing with Tetracycline and Caeni. However, based on the results last week, we decided to do the concentrations below 1 ug/ml. The concentrations are 0 ug/ml, 0.25 ug/ml, 0.5 ug/ml, and 0.75 ug/ml. We expect at least some growth between 0 ug/ml and 0.5 ug/ml. 

Procedure 

To prep, we made four 25ml flasks of normal TGY agar. The reason for this is because we want to ensure that each TGY plates we will be pouring is evenly 25ml. We then recieved our 2.5 ug/ml tetracycline stock from Chad. We also grew our caeni in a 10ml TGY flask. 
To keep in mind, our OD value of caeni was 2.44 after 3-4 days of growth and was diluted to 0.97.

1. We label each of four 25ml flasks of TGY agar their desired tetracycline concentrations and also labeled four empty plates the corresponding concentrations to the flasks. 

               Flask #1 and Plate #1: 0ug/ml 
                Flask #2 and Plate #2: 0.25ug/ml
                Flask #3 and Plate #3: 0.50 ug/ml 
                Flask #4 and Plate #4: 0.75 ug/ml 

2. We heat up the flask one at a time and wait until the TGY agar melted and waited until the flask is LUKEWARM (not baby bottle test because that can still denature our tetracycline due to the extreme heat) to pipette out the excess amount and add in the designated tetracycline amount in ul. See below for amount and concentrations

3. We then vortex the flask for 6-10 seconds to ensure that the tetracycline and media is evenly mixed together. 

4. We gently pour the flask into the corresponding empty plate.

5. While the plate did not solidify yet, we quickly pipette 250 ul of our caeni sample into the plate and using a sterilized loop, we spread the caeni around and we finally wait for the plate to solidify before placing it in the 27 degree Celsius incubator. 

6. Repeat steps 2-5 for the rest of the plates. However, for 0.75 ug/ml, we did it in a flask of 25 ml TGY broth because the fourth agar was contaminated and was placed in the shaking incubator at 30 C. 

Concentrations and Amounts:
0 ug/ml: + and - 0 ul 
0.25 ug/ml: + and - 2.5 ul 
0.50 ug/ml: + and - 5.0 ul 
0.75 ug/ml: + and - 7.5 ul 

Results

After a few days, the results tend to show that there was absolutely no growth in the plates that were more than 0 ug/ml of Tetracycline. 

Figure 1. Tetracycline and Caeni Plate Results






















Figure 2. Gram Stain of Caeni from 0 ug/ml Tetracycline plate



















Conclusion

In conclusion, we can say that since Caeni did not grow at all even in 0.25 ug/ml of Tetracycline, Caeni shows little resistance to Tetracycline itself. Thus, we can say that Caeni's efflux pump allows Tetracycline to enter the cell's membrane and inhibits growth by protein synthesis and shows no strong resistance to pump the Tetracycline out of the cell's membrane before it reaches the cell's proteins. 

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